In this project, we focused our studies on the genetic basis both of orthopoxvirus virulence and of host resistance to virus infection. The acquired knowledge should contribute toward development of safe, effective recombinant vaccinia virus vaccines for animal and human use. A deletion mutant of cowpox virus has been constructed which lacks a functional gene for a 38 kDa protein involved in the generation of a hemorrhagic pock on the chorioallantoic membrane of the chicken embryo. The results to date suggest that the 38 kDa protein directly or indirectly inhibits the generation of chemotactic molecules which are elicited during virus replication in the CAM or, alternatively, blocks the interaction of these molecules with cells of the inflammatory response. Preliminary data suggested that the lipoxygenase pathway of arachidonic acid metabolism may be the target of the 38 kDa protein. The use of various inhibitors of arachidonic acid catabolism have indicated that this fatty acid is required for orthopoxvirus replication and may be necessary directly or indirectly for the assembly of virus specific-membranes. A second orthopoxvirus mutant, which lacked a functional gene encoding ribonucleotide reductase, was shown to be mildly attenuated when injected into BALB/c mice by the intracranial route of inoculation. In vitro and in vivo models are being developed to examine the virus-encoded functions which are important for replication in the epidermis and dermis, and enable the virus to spread from the local site of inoculation.